Abstract Background: Non-alcoholic fatty liver disease (NAFLD) is rapidly turning into the most common liver disorder worldwide. One of the strategies that has been shown to effectively improve NAFLD is regular exercise, which seems to lower steatosis partly independent of weight loss. However, limited data are available about the mechanisms involved. The aim of the present study was to identify the mechanisms underlying the effect of regular exercise on liver steatosis. Methods: Non-obese male mice were rendered steatotic by feeding a sucrose-enriched choline-deficient diet. They were then subjected to daily treadmill running for three weeks, whereas the control mice remained sedentary. Results: Compared to the untrained mice, trained mice showed similar adipose tissue mass but had significantly reduced size of lipid droplets in the liver coupled with a reduction in liver triglyceride content (~30 %, P <0.05). Levels of various plasma lipid parameters and plasma glucose were similar between the trained and untrained mice, whereas levels of hepatic glycogen were significantly higher in the trained mice. Hepatic triglyceride secretion rate and de novo lipogenesis were unchanged between the two sets of mice, as were indicators of lipolysis and autophagy. Finally, whole genome expression profiling indicated a mild stimulatory effect of exercise training on PPARα-mediated regulation of oxidative metabolism, including fatty acid oxidation. Conclusions: Taken together, our study suggests that the lowering of hepatic steatosis by repeated exercise is likely due to activation of fuel oxidation pathways in liver.